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* State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Academia Sinica, Beijing, Peoples Republic of China;
Salk Institute for Biological Studies, La Jolla, California, USA;
Laboratory of Nuclear Analytical Techniques, Institute of High Energy Physics, Academia Sinica;
Institute of Laboratory Animal Science, Chinese Academy of Medical Science;
|| Sychrotron Radiation Laboratory, Institute of High Energy Physics, Academia Sinica; and
¶ Graduate School of the Chinese Academy of Sciences, Beijing, Peoples Republic of China
1Correspondence: Institute of Biophysics, Academia Sinica, 15 Datun Rd., Chaoyang District, Beijing 100101, P.R. China. E-mail: zhaobl{at}sun5.ibp.ac.cn
SPECIFIC AIMS
Accumulative evidence indicates nicotine has therapeutic effect for Alzheimer’s disease (AD), but the mechanism of reducing ßbeta;-amyloidosis by nicotine is not clear. The abnormal interactions of ßbeta;-amyloid (Aßbeta;) with metal ions such as copper and zinc are implicated in Aßbeta; precipitating in AD brains. The original aim of this study was to demonstrate whether nicotine attenuates the Aßbeta; neurotoxicity through regulating copper homeostasis. We used the APPV717I transgenic mice and APPsw-overexpressed cells to investigate the effect of nicotine on the ßbeta;-amyloid neurotoxicity exacerbated by copper.
PRINCIPAL FINDINGS
1. Nicotine decreased the metal levels
(A) Metal element concentration in senile plaque and neuropil
Since nicotine reduces the formation of senile plaques and metals are involved in Aßbeta; precipitation, we measured the effect of nicotine on metal levels in the brain by using synchrotron radiation X-ray fluorescence analysis (SRXRF) analysis. The X-ray spectra collected by SRXRF and the distribution patterns of copper and zinc are shown in Fig. 1
. The copper and zinc concentrations in senile plaques and neuropil were quantified. In Fig. 1B
and C
, data show that the levels of copper and zinc in senile plaques are higher than those in neuropil of both sucrose- and nicotine-treated groups. Furthermore, the levels of copper and zinc are significantly reduced by 
10–20% in senile plaques and neuropil in nicotine-treated mice relative to sucrose-treated controls.
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(B) Effect of nicotine on the metal distribution in the subfield of hippocampal CA1
The distribution of copper and zinc in the subfield of hippocampal CA1 region was also examined (Fig 1E
). Copper is enriched on the pyramidal neuron layer (arrows), whereas the distribution of zinc is relatively spread out. The metal content of either copper or zinc is significantly decreased after nicotine treatment and particularly in the pyramidal neuron layer (arrows).
(C) Relative copper concentration in single cell
According to the method mentioned above, the copper concentrations in the empty vector (neo) and APPsw cells were measured by SRXRF analysis. In neo cell line, both of 10 and 100 µM copper treatment increased the intracellular copper concentration. Nicotine treatment lowered the copper concentration significantly. The intracellular copper concentration in APPsw cells was higher than that in neo cells. Nicotine treatment also decreased the intracellular copper concentration.
2. Nicotine lowered the Aßbeta; positive plaques and decreased the Aßbeta; peptide concentration Aßbeta; peptide secretion and APP expression
The density of Aßbeta; deposition was significantly reduced in the hippocampus of APPV717I transgenic mice treated with nicotine compared with those treated with sucrose alone. The density of senile plaque in the sucrose-treated mice was 3.5%, whereas it was only 1.4% in nicotine-treated mice. Western blotting results showed that the concentration of Aßbeta; peptides reduced 38.2% in the hippocampus and 40.3% in the cortex by the treatment of nicotine relative to the controls.
To detect the effect of nicotine on the APP expression and Aßbeta; secretion, we have stably transfected the SH-SY5Y cell line with vector containing APP, APPsw, and neo. Our data show that copper treatment significantly increases the APP expression in a dose-dependent manner and nicotine administration decreases the APP expression. One main species of ßbeta;-amyloid peptide, Aßbeta;1–40, was investigated using the sandwich ELISA assay kit. The APPsw cell that expresses higher or lower APP concentration received copper or nicotine is also the cell that secretes more or little Aßbeta; peptide; 10 and 100 µM copper treatment increased the Aßbeta; peptide secretion by 20.2 and 28.4%, respectively, whereas the Aßbeta; secretion was decreased after 20 µM nicotine administration.
3. Nicotine reversed the decreasing of cell viability triggered by Aßbeta;
The cell viability was measured by MTT conversion rate, and the results are shown in Fig. 2
. Figure 2A
shows the effect of copper on the cell viability in the neo, APP, and APPsw cell lines. There was no significant difference among the three groups that were not treated by copper and nicotine, whereas if the cells were treated with 10 µM copper, the cell viability of APP and APPsw cells decreased by 11 and 19%, respectively, but there was no effect on neo cell. If the copper concentration was enhanced to 100 µM, the cell viability of neo, APP, and APPSw cells decreased by 25, 38, and 55%, respectively. Figure 2B
depicts that nicotine treatment recovered the APPsw cell viability decreased by copper; 20 µM nicotine administrations increased the cell viability by 41.5 and 75% compared with only 10 and 100 µM copper treatments, respectively.
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4. Nicotine decreased the copper-exacerbated accumulation of reactive oxygen species
Not any significant difference was observed between neo, APP, and APPsw cells without treatment with copper and nicotine. Ten micromolars of copper increased the reactive oxygen species (ROS) generation 27.8% in APP cells and 58.6% in APPsw cells compared with neo cells; 100 µM copper increased the ROS generation 35.4 and 58.2% in APP and APPsw cells, respectively. The ROS accumulation in APPsw cells exacerbated by received 10 and 100 µM copper was decreased 30.3 and 32.8%, respectively, after nicotine administration. In APP cells, the data were 16.5 and 16.8%, respectively.
5. The effect of nicotine on the concentration of copper chaperone for superoxide dismutase and superoxide dismutase-1
In APPsw cells, the ratio of the expression of copper chaperone for superoxide dismutase (CCS for SOD) and SOD-1 (copper/zinc SOD) increased after nicotine treatment. In the hippocampus and cortex of the transgenic mice, the expression of CCS was increased 25 and 67% in the hippocampus and cortex of nicotine-treated mice, respectively, whereas the concentration of SOD1 was lowered 28 and 26% in the hippocampus and cortex, respectively.
CONCLUSIONS
Copper and zinc modulate Aßbeta; aggregation and deposition; therefore, the levels of copper and zinc are crucial to the pathogenesis of AD. We suggest that nicotine reduces ßbeta;-amyloidosis in AD transgenic mice and APPsw-overexpressed cells partly through regulating metal homeostasis since nicotine may regulate metal homeostasis through the following processes (Fig. 3
): 1) nicotine chelates copper and zinc to decrease ROS generation and Aßbeta; aggregation; 2) nicotine affects the copper homeostasis to down-regulate APP expression; and 3) increased expression of CCS reduces the intracellular free copper ions. This result help us better understand the mechanism of nicotine in reducing ßbeta;-amyloidosis.
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ROS production is largely catalyzed by transition metals (especially copper), and oxidative stress plays a critical role in AD pathogenesis. In the present study, the association of metal levels and Aßbeta; toxicity is demonstrated in three respects: 1) the effect on cell viability by metal alone and in the combination with APP and Aßbeta;; 2) Aßbeta;-induced neurotoxicity relevant to oxidative stress indicated by ROS production; and 3) APPsw cells expressed APP and generated Aßbeta;, so that Aßbeta;Cu2+ and APPCu2+ can catalyze more ROS generation than APP cells that only expressed APP.
Nicotine shows protective effects against Aßbeta;-induced neurotoxicity in vivo and in vitro. Although nicotine acts on nAChRs, it also contains metal chelating abilities. It has been shown that maternal nicotine exposure resulted in a reduction of the copper content in a neonatal lung. In addition, evidence has been accumulated that nicotine might chelate metals through pyridine nitrogen. Indeed, nicotine reduces the levels of copper and zinc in senile plaque and neuropil (Fig. 1)
, which counteracts the morbid metal accumulation.
The brain regional and the intracellular copper concentrations regulated by nicotine were further supported by the changes of both CCS and SOD1 levels. CCS directly inserts copper into SOD1 and prevents the accumulation of free copper ions in cells. In the transgenic mice brain, our data suggest that an increase of CCS expression by nicotine causes a decrease in free intracellular copper levels, since more expression of CCS transfers more free intracellular copper ions into proteins. Therefore, the copper ions that can be transported to the extracellular space are reduced, which results in an inhibition of Aßbeta; aggregation mediated by free copper ions.
Although nicotine has been proven to have beneficial effect in neurodegenerative disease, the exactly mechanism is still unclear. Our data suggest the novel idea that nicotine may regulate the metal homeostasis. A better understanding of this mechanism of nicotine may conduce to our better understandings on the mechanism of nicotine in reducing ßbeta;-amyloidosis.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-5214fje
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